LCMS characteristic chromatogram of tortoise-shell glue and preparation thereof as well as establishment method and application of LCMS characteristic chromatogram

Abstract

The invention belongs to the technical field of detection, and particularly relates to an LCMS (Liquid Chromatography Mass Spectrometry) characteristic chromatogram of tortoise-shell glue and a preparation thereof as well as an establishment method and application of the LCMS characteristic chromatogram of the tortoise-shell glue and the preparation thereof. Clear and detailed LCMS characteristic spectrums of the tortoise-shell glue and preparations thereof can be established according to contour mass spectrum information of the tortoise-shell glue, overall analysis of multiple characteristic polypeptide components is realized, and the limitation that analysis is only carried out in a single extraction ion flow channel in an existing method is solved; the obtained LCMS characteristic chromatogram of the tortoise-shell glue and the preparation thereof can effectively reflect the integrity and complexity of polypeptide components of the tortoise-shell glue and the preparation thereof, so that the anti-counterfeiting products of the tortoise-shell glue and the preparation thereof can be effectively distinguished, the quality of the tortoise-shell glue and the preparation thereof is comprehensively evaluated and controlled, and a guarantee is provided for clinical safe and effective medication.

Description

LCMS Characteristic Chromatography of Tortoise Shell Glue and its Preparations, Its Establishment Method and Application Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to the LCMS characteristic spectrum of tortoise shell glue and its preparations, its establishment method, and its application. Background Technology

[0002] Tortoise shell glue is one of the most precious medicinal materials in traditional Chinese medicine, alongside ginseng, deer antler, and donkey-hide gelatin. Its application history is long, dating back to the Han Dynasty. It has always been highly regarded by physicians as an essential medicine for nourishing yin, stopping bleeding, and tonifying the kidneys, and is widely used in various clinical departments. Classic medical texts such as the *Compendium of Materia Medica* record its effects of nourishing yin and suppressing yang, tonifying the kidneys and strengthening bones, and nourishing blood and the heart. Modern pharmacological research and clinical practice have shown that tortoise shell glue has good potential in treating autoimmune diseases, osteoporosis, and cardiovascular and nervous system diseases. This indicates that tortoise shell glue is not only a good medicine for nourishing yin, but also a traditional Chinese medicine that integrates prevention, treatment, and rehabilitation functions, making it an important part of the treasure trove of Chinese medicine. With the booming development of the health industry and the increasing global influence of traditional Chinese medicine, tortoise shell glue is benefiting more people at home and abroad with its unique medicinal value. Tortoise shell glue is a gelatinous traditional Chinese medicine made primarily from the shells of turtles. Its material basis is complex, mainly containing proteins, polypeptides, and trace elements.

[0003] Based on the above reasons, establishing an effective quality control method for tortoise shell glue is the key to ensuring its clinical efficacy and safety. However, the current quality control of tortoise shell glue mainly faces the following challenges: (1) Prominent adulteration problem: Due to the scarcity and high cost of tortoise shell raw materials, there are cases of adulteration or counterfeiting using other animal skin sources such as cowhide and pigskin in the market. These counterfeit products are not only ineffective, but also pose safety risks. (2) Limitations of existing detection methods: Although the Chinese Pharmacopoeia includes identification methods based on characteristic peptides, existing technologies mostly use conventional high-performance liquid chromatography-mass spectrometry for identification. However, this identification method has problems such as long analysis time (e.g., up to 40 minutes) and low efficiency. More importantly, most methods cannot simultaneously complete the identification of genuine and counterfeit tortoise shell glue and the detection of illegal addition of various common miscellaneous skin sources (such as cowhide and pigskin). Each experiment can only complete the detection of a single target, making it difficult to comprehensively monitor the quality. (3) Insufficient means of overall quality evaluation: The proteins in tortoise shell glue undergo dynamic changes during processing, and the molecular weight distribution of proteins in the final product is also significantly different from other gelatinous medicinal materials (such as donkey-hide gelatin and deer antler glue). However, current analytical methods based on mass spectrometry are mostly limited to qualitative and quantitative analysis of single or a few characteristic peptides. This "single index" or "limited index" approach makes it difficult to comprehensively capture and characterize the overall features of the complex polypeptide system of tortoise shell glue, thus limiting the development of characteristic spectral technologies that can fully reflect its intrinsic quality.

[0004] Currently, although some studies have used liquid chromatography-mass spectrometry (LC-MS) to detect characteristic peptides and adulterants in tortoise shell glue, and some studies have used ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) and metabolomics to compare and analyze the compound differences of different types of glue-based traditional Chinese medicines, there is still a lack of systematic research both domestically and internationally that uses the tortoise shell glue polypeptide as a whole as a material basis to establish its exclusive liquid chromatography-mass spectrometry (LC-MS) characteristic spectrum.

[0005] Chinese invention patent publication CN116818921A discloses a method for identifying tortoise shell glue and simultaneously detecting off-derived components. This method utilizes ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) to achieve the identification of tortoise shell glue and the simultaneous detection of off-derived components. However, the conditions set in this method need improvement, and no effective characteristic spectra are provided.

[0006] Therefore, there is an urgent need to establish a new LCMS characteristic chromatographic method for tortoise shell glue and its preparations. This method is expected to effectively reflect the integrity and complexity of the polypeptide components of tortoise shell glue, overcome the limitations of existing detection methods in terms of efficiency and throughput, and thus achieve comprehensive, scientific evaluation and precise control of the quality of tortoise shell glue and its preparations, laying a solid foundation for ensuring the safety and effectiveness of clinical medication. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for establishing LCMS characteristic spectra of tortoise shell glue and its preparations. This method enables the overall detection of peptides in tortoise shell glue and its preparations, effectively supplementing existing quality control systems and providing a reliable basis for the identification and quality control of tortoise shell glue and its preparations.

[0008] Specifically, this is achieved through the following technical means: A method for establishing LCMS characteristic spectra of tortoise shell glue and its preparations, comprising the following steps: 1) mixing tortoise shell glue and its preparations with ammonium bicarbonate solution and subjecting them to ultrasonic treatment, then adding trypsin for enzymatic hydrolysis to prepare a test solution; preparing a reference solution of tortoise shell glue reference material; and preparing a reference solution of tortoise shell polypeptide C1 with ammonium bicarbonate.

[0009] 2) The test solution, reference medicinal material solution and reference standard solution obtained in step 1) were detected by liquid chromatography-mass spectrometry to obtain the LCMS characteristic chromatograms of tortoise shell glue and its preparations.

[0010] Preferably, in step 1), the preparation of the reference drug solution from the tortoise shell glue reference material specifically involves: mixing the tortoise shell glue reference material with ammonium bicarbonate and sonicating it, adding trypsin for enzymatic hydrolysis, filtering, and taking the filtrate to obtain the reference drug solution.

[0011] Preferably, in step 1), the mass-to-volume ratio of tortoise shell glue reference material to ammonium bicarbonate solution, and the mass-to-volume ratio of tortoise shell glue and its preparation to ammonium bicarbonate solution are both 1g:(200~1000)ml, the mass ratio of trypsin to tortoise shell glue and its preparation is 1:(2~4), and the concentration of trypsin is 0.5~2mg / ml.

[0012] Preferably, the ultrasonic treatment power is 300-500W, the ultrasonic treatment frequency is 30-50kHz, and the ultrasonic treatment time is 10-40min.

[0013] Preferably, in step 1), the mass concentration of turtle-derived polypeptide C1 in the reference solution is 1-5 μg / ml.

[0014] Preferably, in step 2), the chromatographic conditions of the liquid chromatography-mass spectrometry method are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; mobile phase A is 0.08-0.12 wt.% formic acid, and mobile phase B is acetonitrile, with the mobile phase A and mobile phase B eluted in a gradient ratio by volume.

[0015] Preferably, the gradient elution time is 20 minutes, and during the period from 0 to 12 minutes, the volume ratio of mobile phase A gradually increases from 2% to 10%, and the volume ratio of mobile phase B gradually decreases from 98% to 90%; during the period from 12 to 20 minutes, the volume ratio of mobile phase A gradually increases from 10% to 25%, and the volume ratio of mobile phase B gradually decreases from 90% to 75%.

[0016] Preferably, the chromatographic column is a chromatographic column with an inner diameter of 2.1 mm, a length of 100 mm, and a packing particle size of 1.7 μm; or a chromatographic column with an inner diameter of 2.1 mm, a length of 100 mm, and a packing particle size of 1.8 μm.

[0017] Further preferably, the chromatographic column comprises a Thermo Syncronis C18 column, 2.1 mm × 100 mm, 1.7 μm; or a Waters BEH C18 column, 2.1 mm × 100 mm, 1.7 μm; or a MicroPulite HSS T3 column, 2.1 mm × 100 mm, 1.8 μm.

[0018] Preferably, in the liquid chromatography-mass spectrometry method, the flow rate of the mobile phase is 0.1-0.3 mL per minute, the column temperature is 35-45℃, and the theoretical plate number calculated based on the C1 peak of the turtle-derived polypeptide is not less than 5000.

[0019] Preferably, in step 2), the ion pairs in the liquid chromatography-mass spectrometry (LC-MS) are set to the following mass-to-charge ratios for the parent ion and daughter ion: Peak 1 is m / z 427.2 (double charge) → 127.1 and m / z 427.2 (double charge) → 699.3; Peak 2 is m / z 523.8 (double charge) → 619.3 and m / z 523.8 (double charge) → 876.4; Peak 3 is m / z 568.7 (double charge) → 724.3 and m / z 568.7 (double charge) → 852.0; Peak 4... The seven ion pairs are: m / z 441.7 (double charge) → 528.3 and m / z 441.7 (double charge) → 629.3, peak 5 is m / z 626.3 (double charge) → 696.4 and m / z 626.3 (double charge) → 753.4, peak 6 is m / z 631.3 (double charge) → 546.4 and m / z 631.3 (double charge) → 921.4, and peak 7 is m / z 758.3 (double charge) → 615.1 and m / z 758.3 (double charge) → 544.1.

[0020] LCMS characteristic spectrum of tortoise shell glue and its preparation, wherein the characteristic spectrum is obtained by the above-mentioned LC-MS method, the characteristic spectrum uses the tortoise-derived polypeptide C with an ion mass-to-charge ratio of m / z 631.3 as the reference peak S, and has at least 6 characteristic peaks; the ion mass-to-charge ratios of the 6 characteristic peaks are m / z 427.2, m / z 523.8, m / z 568.7, m / z 441.7, m / z 626.3 and m / z 758.3, respectively, and the relative retention times of the characteristic peaks are all within ±15% of their respective specified values, the specified values ​​being 0.39, 0.42, 0.56, 0.58, 0.66 and 1.19, respectively.

[0021] Preferably, the characteristic spectrum exhibits at least seven characteristic peaks: peak 1 has a mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.39 ± 15%; peak 2 has a mass-to-charge ratio of m / z 523.8 and a relative retention time of 0.42 ± 15%; peak 3 has a mass-to-charge ratio of m / z 568.7 and a relative retention time of 0.56 ± 15%; peak 4 has a mass-to-charge ratio of m / z 441.7 and a relative retention time of 0.58 ± 15%; peak 5 has a mass-to-charge ratio of m / z 626.3 and a relative retention time of 0.66 ± 15%; peak 6 is turtle-derived polypeptide C at m / z 631.3, peak 6 is a reference peak S; and peak 7 has a mass-to-charge ratio of m / z 758.3 and a relative retention time of 1.19 ± 15%.

[0022] Preferably, the amino acid sequence of peak 1 is GPSGPEGPR, the amino acid sequence of peak 2 is GLAGEPPGSAGSK, the amino acid sequence of peak 3 is GVQGAPGPQGPR, the amino acid sequence of peak 4 is VGPTGPAGAR, the amino acid sequence of peak 5 is GESGPAGPAGPAGAR, the amino acid sequence of peak 6 is GIPGPAGPAGAAGPR, and the amino acid sequence of peak 7 is GDGGPPGITGFPGASGR.

[0023] An application of LCMS feature map in the detection of tortoise shell glue, wherein the LCMS feature map is the LCMS feature map described above or the LCMS feature map obtained by the above method.

[0024] The present invention achieves the following beneficial effects: 1. By setting specific steps and specific chromatographic and mass spectrometric conditions, especially specific gradient elution conditions and specific settings for multiple ion pairs, the present invention can establish clear and detailed LCMS characteristic spectra of tortoise shell glue and its preparations based on the profile mass spectrometry information of tortoise shell glue, realizing the overall analysis of multiple characteristic polypeptide components, and overcoming the limitation of existing methods that only perform analysis within a single extraction ion current channel. The obtained LCMS characteristic spectra of tortoise shell glue and its preparations can effectively reflect the integrity and complexity of the polypeptide components of tortoise shell glue and its preparations, thereby effectively distinguishing counterfeit tortoise shell glue and its preparations, comprehensively evaluating and controlling the quality of tortoise shell glue and its preparations, and thus providing a guarantee for safe and effective clinical use.

[0025] 2. The technical solution of this invention successfully established an LC-MS characteristic chromatographic method for tortoise shell glue and its preparations. Firstly, methodological validation showed that the method exhibited good specificity, precision, repeatability, and stability (RSD < 2.0%). Secondly, analysis of 17 batches of qualified tortoise shell glue identified 7 common peak patterns with peak 6 as the reference peak. The batch-to-batch similarity was greater than 0.90, and the relative retention time fluctuation was within ±15%, indicating good chromatographic reproducibility. The characteristic chromatograms obtained based on the method of this invention can be effectively used for the detection of tortoise shell glue-related products. Furthermore, through specific settings of the detection method, the detection method based on the characteristic chromatograms of this invention is simple to operate, highly stable, and has good reproducibility. The chromatograms developed in this invention possess strong specificity, clearly distinguishing common and easily confused products such as turtle shell glue, yellow gelatin, donkey-hide gelatin, deer antler glue, horse hide glue, and new donkey-hide gelatin (as shown in Figures 11-16). The characteristic peaks of the chromatograms obtained by the method established in this invention are comprehensively displayed. By comparing the common peaks of standard fingerprint chromatograms, rapid identification and control of turtle shell glue products such as turtle shell glue, turtle shell glue formula granules, and turtle shell glue compound preparations can be achieved. This effectively distinguishes counterfeit turtle shell glue products, facilitating a comprehensive assessment of the intrinsic quality of medicinal materials and processed medicinal slices, and ensuring the safety and efficacy of clinical medication. The LC-MS characteristic chromatogram method established in this invention is stable and reliable. The constructed turtle shell glue characteristic chromatograms are highly specific and reproducible, providing a scientific and efficient analytical method and data support for the quality consistency control, authenticity identification, and subsequent quality standard improvement of turtle shell glue and its preparations. Attached Figure Description

[0026] Figure 1 is a characteristic spectrum of the tortoise shell glue of the present invention.

[0027] Figure 2 shows the characteristic spectra of 17 batches of tortoise shell glue samples in one embodiment of the present invention.

[0028] Figure 3 is a chromatogram of a tortoise shell glue test sample according to one embodiment of the present invention.

[0029] Figure 4 is a negative control chromatogram of one embodiment of the present invention.

[0030] Figure 5 is a chromatogram of instrument precision according to one embodiment of the present invention.

[0031] Figure 6 is a repeatability chromatogram of one embodiment of the present invention.

[0032] Figure 7 is an intermediate precision chromatogram of one embodiment of the present invention.

[0033] Figure 8 is a stability chromatogram of one embodiment of the present invention.

[0034] Figure 9 is a chromatogram of the tortoise shell glue formulation particles according to another embodiment of the present invention.

[0035] Figure 10 is a chromatogram of a tortoise shell glue test sample according to another embodiment of the present invention.

[0036] Figure 11 is a chromatogram of a yellow gelatin sample according to another embodiment of the present invention.

[0037] Figure 12 is a chromatogram of a donkey-hide gelatin test sample according to another embodiment of the present invention.

[0038] Figure 13 is a chromatogram of a deer antler glue test sample according to another embodiment of the present invention.

[0039] Figure 14 is a chromatogram of a turtle shell gelatin sample according to another embodiment of the present invention.

[0040] Figure 15 is a chromatogram of a horsehide glue test sample according to another embodiment of the present invention.

[0041] Figure 16 is a chromatogram of a novel donkey-hide gelatin test sample according to another embodiment of the present invention. Detailed Implementation

[0042] The embodiments of the present invention will now be clearly and completely described in conjunction with examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the instruments used in the embodiments of this invention are as follows: the mass spectrometer is a Waters XevoTQ-S micro triple quadrupole mass spectrometer; the chromatograph is a Waters ACQUITY UPLC H-Class high-performance liquid chromatograph; the ultrasonic instrument is a KQ-500DE ultrasonic instrument from Kunshan Ultrasonic Instrument Co., Ltd.; the balance is a 0.01% balance from Fujian Huazhi PTX-FA210S and a 0.01% balance from Mettler Toledo ME155DU; the water bath is a commercially available ordinary water bath; and the drying oven is a commercially available ordinary drying oven. The samples used in this embodiment are as follows: the test sample is 17 batches of tortoise shell glue samples collected from the market (these are tortoise shell glue samples that have passed testing); the tortoise shell glue reference material is purchased from the China National Institutes for Food and Drug Control, batch number: 121693-201702; and the tortoise-derived polypeptide C is purchased from Qiangyao Biotechnology Co., Ltd., batch number: 04010096129, with a purity of 96.5%. Acetonitrile and formic acid were commercially available chromatographic grade, water was purified water, and the remaining reagents were analytical grade.

[0044] Example 1 This example illustrates a methodological evaluation of a feature map (including specificity evaluation, instrument precision testing, method repeatability testing, intermediate precision testing, and stability testing) (passing the evaluation indicates that the method has been successfully established).

[0045] 1. First, specificity investigation is conducted, including the following steps: 1) Preparation of blank sample: Accurately measure 1 ml of 1 wt.% ammonium bicarbonate solution into a 5 ml volumetric flask, and add 1 ml of trypsin solution to the 5 ml volumetric flask; the trypsin solution is freshly prepared before use, and the preparation process is as follows: take sequence analysis grade trypsin, mix the sequence analysis grade trypsin with 1 wt.% ammonium bicarbonate solution to prepare a trypsin solution containing 1 mg of sequence analysis grade trypsin per 1 ml of mixture; then add 1 wt.% ammonium bicarbonate solution to the 5 ml volumetric flask, the amount added is enough to dilute the mixture to the mark (i.e., 5 ml), and shake well, maintain the temperature at 37℃ for 12 hours for enzymatic hydrolysis, and then filter the material in the 5 ml volumetric flask, and take the filtrate to obtain the blank sample solution (i.e., negative control solution).

[0046] 1 μL of the prepared blank sample solution and 1 μL of the test solution from Example 1 were precisely pipetted into the liquid chromatograph. The results showed negative results with no interference, indicating that the specificity test was successful.

[0047] 2. Next, the instrument precision test is carried out, including the following steps: Take the same sample solution, inject it 6 times, and measure the relative retention time of the 7 common peaks. The data results are shown in Table 1. Table 1 shows the relative retention time results of the instrument precision for 6 repeated injections.

[0048] Table 1 The RSD (%) in Table 1 represents the relative standard deviation. As shown in Table 1, the relative retention times of each characteristic peak and the reference S peak in the 6 injections are all less than 2.0%, indicating that the instrument precision is good.

[0049] 3. Next, the method repeatability test is carried out, including the following steps: take 6 samples of the same batch number, measure the relative retention time of 7 common peaks, and obtain the results shown in Table 2. Table 2 is a data table of repeatability relative retention time results.

[0050] Table 2 The results in Table 2 show that the relative retention times of each characteristic peak and the reference peak S are all less than 2.0%, indicating that the method has good repeatability.

[0051] 4. Next, the intermediate precision was investigated by different operators, including the following steps: In this investigation, three inspectors measured the same batch of tortoise shell glue at different times using the same equipment. The relative retention time results are shown in Table 3. Table 3 is the data table of intermediate precision relative retention time results.

[0052] Table 3 As shown in Table 3, the relative average deviation of the relative retention times of each characteristic peak and the reference peak obtained by the three staff members using the same equipment at different times is less than 2.0%, indicating that the intermediate precision of this method is good.

[0053] 5. Next, stability studies were conducted, including the following steps: Samples of the same batch were injected at 0, 4, 8, 12, 24, 36, and 48 hours, and the relative retention times of the seven common peaks were measured. The results are shown in Table 4. Table 4 shows the relative retention time results for stability.

[0054] Table 4 As shown in Table 4, the relative retention times of all characteristic peaks of all samples from 0 hours to 48 hours with the reference peak are all less than 2.0%. This result indicates that the relative retention times of all characteristic peaks of the test solution are stable within 48 hours, which meets the determination requirements.

[0055] Example 2 This example demonstrates that by analyzing 17 batches of tortoise shell glue, calculating the batch-to-batch similarity and relative retention time fluctuation, it shows that the chromatogram reproducibility is good. This example includes the following steps: 1) Preparation of reference solution, reference medicinal material reference solution, and test solution: 2 mg of the tortoise-derived polypeptide C reference standard was accurately weighed using a 1 / 100,000 balance, and then added to 1000 ml of ammonium bicarbonate solution with a mass percentage concentration of 1 wt.% and mixed to obtain a mixed solution containing 2.0 μg of tortoise-derived polypeptide C per 1 ml of ammonium bicarbonate solution, which was used as the reference solution.

[0056] Weigh 0.1g of the tortoise shell glue reference medicine powder using a 0.01g balance. Place the weighed tortoise shell glue reference medicine in a 50ml volumetric flask. Add 40ml of 1wt.% ammonium bicarbonate solution to the volumetric flask to obtain a mixture. Sonicate the mixture for 30 minutes. Then, continue to add 1wt.% ammonium bicarbonate solution to dilute to the mark (50ml). Shake well to obtain a diluted mixture. Accurately measure 1 ml of the diluted mixture and place it into a 5 ml volumetric flask; take sequence-analytical grade trypsin and mix it with 1 wt.% ammonium bicarbonate solution to prepare a trypsin solution containing 1 mg of sequence-analytical grade trypsin per 1 ml of the mixture; add 1 ml of the trypsin solution to the 5 ml volumetric flask, then add 1 wt.% ammonium bicarbonate solution to the 5 ml volumetric flask to dilute to the mark (5 ml), and shake well. Maintain the enzymatic hydrolysis at a constant temperature of 37°C for 12 hours, then filter the material in the 5 ml volumetric flask, and take the filtrate as the reference solution for the control drug.

[0057] Weigh 0.1 g (100 mesh) of the test sample powder using a 0.01 g balance. Place the test sample in a 50 ml volumetric flask and add 40 ml of 1 wt.% ammonium bicarbonate solution to the 50 ml volumetric flask to obtain a mixed solution. Sonicate the mixed solution at a power of 250 W and a frequency of 40 kHz for 30 minutes. Then, add 1 wt.% ammonium bicarbonate solution to the 50 ml volumetric flask to dilute to the mark (50 ml) and shake well to obtain a diluted solution. Then, accurately measure 1 ml of the obtained diluted solution and transfer it to a 5 ml volumetric flask. Then, add 1 ml of trypsin solution to the 5 ml volumetric flask. The trypsin solution is freshly prepared before use. The preparation process is as follows: take sequence analysis grade trypsin, mix the sequence analysis grade trypsin with 1 wt.% ammonium bicarbonate solution to prepare a trypsin solution containing 1 mg of sequence analysis grade trypsin per 1 ml of mixture. Then, add 1 wt.% ammonium bicarbonate solution to the 5 ml volumetric flask to dilute to the mark (5 ml), shake well, and maintain the enzymatic hydrolysis at a constant temperature of 37℃ for 12 hours. Then, filter the material in the 5 ml volumetric flask and take the filtrate as the test solution.

[0058] 2) Setting up chromatographic and mass spectrometry conditions: A chromatographic column was set up using octadecylsilane-bonded silica gel as the packing material. The column length was 100 mm, and the inner diameter was 2.1 mm (in different embodiments, the column could be Thermo Syncronis C18, 2.1 mm × 100 mm, 1.7 μm; or Waters BEH C18, 2.1 mm × 100 mm, 1.7 μm; or MicroPulite HSS T3, 2.1 mm × 100 mm, 1.8 μm), with a packed particle size of 1.7 μm. Acetonitrile was used as mobile phase A, and 0.1 wt.% formic acid aqueous solution was used as mobile phase B. Gradient elution was performed at different time periods according to the settings in Table 5, with a flow rate of 0.3 ml / min and a column temperature of 40 °C. Table 5 shows the mobile phase setup for Example 1.

[0059] Table 5 Mass spectrometry analysis was performed using a triple quadrupole mass spectrometer detector and multiple reaction monitoring (MRM) was performed in positive ion mode with electrospray ionization (ESI). The specific settings for the monitored ion pairs are shown in Table 6. Table 6 is a table of settings for peaks and parent and daughter ion pairs in mass spectrometry analysis.

[0060] Table 6 Take the reference solution of the aforementioned medicinal material, inject 1 μl, and the signal-to-noise ratio of the MRM chromatographic peaks determined according to the above detection ion pairs is greater than 3:1.

[0061] 3) Establishment of characteristic chromatograms: Data were collected on the reference solutions of the reference medicinal materials and reference standards according to the chromatographic and mass spectrometric conditions in step 2) to obtain the total ion chromatogram (TIC) and the extracted ion chromatograms (XIC) of each channel of the tortoise shell glue sample.

[0062] Following the chromatographic and mass spectrometric conditions set in step 2), 17 batches of tortoise shell glue samples were analyzed, and LCMS spectra of 17 sample solutions were obtained. The raw data were exported and imported into the chempattern software. A profile mass spectrum was selected, with a mass-to-nuclear ratio tolerance of 0.01 Da. A common pattern was generated using Gaussian curve simulation as the standard characteristic spectrum. The characteristic spectrum of tortoise shell glue shown in Figure 1 was obtained. Peak 6 corresponds to the retention time of the tortoise shell polypeptide C reference peak. The corresponding peak of the tortoise shell polypeptide C reference peak was taken as the S peak (i.e., peak 6 is the S peak). The relative retention times of peaks 1 to 5, peak 7 and the S peak were calculated and set within ±15% of the specified values. The specified values ​​are: 0.39 (peak 1), 0.42 (peak 2), 0.56 (peak 3), 0.58 (peak 4), 0.66 (peak 5), and 1.19 (peak 7).

[0063] The TIC spectra of the test solutions from 17 batches of tortoise shell glue were exported, and the relative retention times of the common peaks of the samples were obtained as shown in Table 7. Table 7 is a table of relative retention time data for 17 batches of tortoise shell glue samples.

[0064] Table 7 The characteristic spectra of 17 batches of tortoise shell glue are shown in Figure 2.

[0065] Table 7 and Figure 2 show that, through the analysis of 17 batches of qualified tortoise shell glue, 7 common peak patterns with peak 6 as the reference peak were identified. The similarity between batches was greater than 0.90, and the relative retention time fluctuation was within ±15%, indicating good reproducibility of the spectrum.

[0066] Example 3 This example demonstrates the determination of the characteristic spectrum of tortoise shell glue formula granules based on the characteristic spectrum obtained in Example 2. The instrument used in this example is exactly the same as that used in Example 1. The sample used in this example is: one batch of tortoise shell glue formula granules purchased from the market; the remaining tortoise shell glue reference material, tortoise-derived polypeptide C, acetonitrile, formic acid, water, and other reagents are the same as in Example 2.

[0067] This embodiment includes the following steps: 1) Preparation of reference solution, reference medicinal material solution and test solution: The preparation methods of reference solution and reference medicinal material solution are the same as in Example 2.

[0068] The preparation process of the test solution is as follows: Weigh 0.1g of the tortoise shell glue formula granules using a 0.01g balance, place the sample to be tested in a 50ml volumetric flask, add 40ml of 1wt.% ammonium bicarbonate solution to the 50ml volumetric flask to obtain a mixed solution, subject the mixed solution to ultrasonic treatment at a power of 250W and a frequency of 40kHz for 30 minutes, add 1wt.% ammonium bicarbonate solution to the 50ml volumetric flask to dilute to the mark (50ml), shake well to obtain a diluted solution. Then, accurately measure 1 ml of the diluted solution and transfer it to a 5 ml volumetric flask. Then, add 1 ml of trypsin solution to the 5 ml volumetric flask. The trypsin solution is freshly prepared before use. The preparation process is as follows: take sequence analysis grade trypsin, mix the sequence analysis grade trypsin with 1 wt.% ammonium bicarbonate solution to prepare a trypsin solution containing 1 mg of sequence analysis grade trypsin per 1 ml of mixture. Then, add 1 wt.% ammonium bicarbonate solution to the 5 ml volumetric flask to dilute to the mark (5 ml), shake well, and maintain the enzymatic hydrolysis at a constant temperature of 37°C for 12 hours. Then, filter the material in the 5 ml volumetric flask, and take the filtrate as the test solution.

[0069] 2) Setting up chromatographic and mass spectrometry conditions: The chromatographic column was set up with octadecylsilane-bonded silica gel as the packing material. The column length was 100 mm, the inner diameter was 2.1 mm, and the packing particle size was 1.7 μm. Acetonitrile was used as mobile phase A, and 0.1 wt.% formic acid aqueous solution was used as mobile phase B. Gradient elution was performed by setting the ratio of mobile phases at different time periods according to the settings in Table 5 of Example 1 above. The flow rate was 0.3 ml / min, and the column temperature was set to 40 °C.

[0070] Mass spectrometry analysis was performed using a triple quadrupole mass spectrometer detector, and multiple reaction monitoring (MRM) was performed in positive ion mode using electrospray ionization (ESI). The specific settings for the monitored ion pairs are shown in Table 6 of Example 2.

[0071] Take the reference solution of the reference medicinal material, inject 1 μl, and the signal-to-noise ratio of the MRM chromatographic peaks determined according to the above detection ion pairs is greater than 3:1.

[0072] 3) Establishment of Characteristic Chromatography: Following the chromatographic and mass spectrometric conditions set in step 2), the tortoise shell glue formulation granule sample was analyzed. The characteristic chromatography obtained in Example 2 was compared with that in Figure 9. A comparison between Figure 9 and Figure 1 shows that the peak distribution patterns of the two LCMS characteristic chromatograms are highly consistent, indicating stable chromatographic separation conditions and unchanged component polarity order. Differences in peak intensity may originate from minor fluctuations in sample concentration, injection volume, or detector response. However, the retention times of each peak in Figures 1 and 9 are consistent, thus not affecting the qualitative analysis results. This verifies that the tortoise shell glue formulation granules contain genuine tortoise shell glue. The consistency of the tortoise shell glue formulation granules also proves that the LCMS characteristic chromatogram established in this invention is correct and can withstand verification.

[0073] Example 4 illustrates the determination of the characteristic spectrum of Guanhuangmu Granules, a compound preparation containing tortoise shell glue, based on the characteristic spectrum obtained in Example 2. Guanhuangmu Granules contain tortoise shell glue. Using the same characteristic spectrum determination method described in Example 3 of this invention, Guanhuangmu Granules were used as the test solution, and the same steps and methods as in Example 3 were performed. The results showed that the retention time, relative retention time, and mass spectrometry information of each characteristic peak in the characteristic spectrum were consistent with the characteristic spectra established in Examples 2 (tortoise shell glue) and 3 (tortoise shell glue formulation granules) of this invention. This indicates that the method can effectively identify and detect the characteristic polypeptide components from tortoise shell glue in the complex matrix of Guanhuangmu Granules. This method can be used for the authenticity identification of tortoise shell glue in Guanhuangmu Granules and the evaluation of overall quality consistency.

[0074] Example 5 This example illustrates a verification test example of differentiating related adhesive varieties based on the feature spectrum obtained in Example 2.

[0075] The instruments used in this embodiment are exactly the same as those used in Example 2. The samples used in this embodiment are: tortoise shell glue, yellow gelatin, donkey-hide gelatin, deer antler glue, turtle shell glue, horse skin glue, and new donkey-hide gelatin, all collected and purchased from the market; the remaining tortoise shell glue reference materials, tortoise-derived polypeptide C, acetonitrile, formic acid, water, and other reagents are the same as in Example 2.

[0076] This embodiment includes the following steps: 1) Preparation of reference solution, reference medicinal material solution and test solution: The preparation methods of reference solution and reference medicinal material solution are the same as in Example 2.

[0077] The preparation process of the test solution is as follows: Weigh 0.1g of the powders of the yellow gelatin, donkey-hide gelatin, deer antler gelatin, turtle shell gelatin, horse skin gelatin, and new donkey-hide gelatin using a 0.01g balance. Place the test sample in a 50ml volumetric flask and add 40ml of 1wt.% ammonium bicarbonate solution to the 50ml volumetric flask to obtain a mixed solution. Sonicate the mixed solution at a power of 250W and a frequency of 40kHz for 30 minutes. After sonication, add 1wt.% ammonium bicarbonate solution to the 50ml volumetric flask to dilute to the mark (50ml). Shake well to obtain the diluted solution. Then, accurately measure 1 ml of the diluted solution and transfer it to a 5 ml volumetric flask. Then, add 1 ml of trypsin solution to the 5 ml volumetric flask. The trypsin solution is freshly prepared before use. The preparation process is as follows: take sequence analysis grade trypsin, mix the sequence analysis grade trypsin with 1 wt.% ammonium bicarbonate solution to prepare a trypsin solution containing 1 mg of sequence analysis grade trypsin per 1 ml of mixture. Then, add 1 wt.% ammonium bicarbonate solution to the 5 ml volumetric flask to dilute to the mark (5 ml), shake well, and maintain the enzymatic hydrolysis at a constant temperature of 37°C for 12 hours. Then, filter the material in the 5 ml volumetric flask, and take the filtrate as the test solution.

[0078] 2) Set the chromatographic and mass spectrometry conditions: use octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm); use acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, and perform gradient elution according to the specifications in the table below, with a flow rate of 0.3 ml per minute and a column temperature of 40 °C.

[0079] Mass spectrometry analysis was performed using a triple quadrupole mass spectrometer detector, and multiple reaction monitoring (MRM) was performed in positive ion mode using electrospray ionization (ESI). The specific settings for the monitored ion pairs are shown in Table 6 of Example 2.

[0080] 3) Comparison of characteristic spectra: The characteristic spectra of tortoise shell glue, yellow gelatin, donkey-hide gelatin, deer antler glue, turtle shell glue, horse skin glue, and new donkey-hide gelatin were obtained by establishing the characteristic spectra in the same way as in Example 3. As can be seen from the results shown in Figures 10 to 16, the characteristic peptide spectra of yellow gelatin, donkey-hide gelatin, deer antler glue, turtle shell glue, horse skin glue, and new donkey-hide gelatin are completely different from the characteristic peptide spectra of tortoise shell glue obtained in Example 2 of this invention. This proves that the characteristic peptide spectra of tortoise shell glue set by this invention can distinguish tortoise shell glue from other glue varieties such as turtle shell glue, yellow gelatin, donkey-hide gelatin, deer antler glue, new donkey-hide gelatin, and horse skin glue, thereby enabling the identification of the above-mentioned materials that are mixed in.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing LCMS characteristic spectra of tortoise shell glue and its preparations, characterized in that, The procedure includes the following steps: 1) Mixing tortoise shell glue and its preparations with ammonium bicarbonate solution and sonicating them, then adding trypsin for enzymatic hydrolysis to prepare a test solution; preparing a reference solution of tortoise shell glue reference material, and mixing tortoise-derived polypeptide C1 with ammonium bicarbonate to prepare a reference solution of reference standard; 2) Detecting the test solution, reference solution of reference material, and reference solution of reference standard obtained in step 1) using liquid chromatography-mass spectrometry to obtain the LCMS characteristic chromatograms of tortoise shell glue and its preparations.

2. The method for establishing the LCMS characteristic spectrum of tortoise shell glue and its preparations according to claim 1, characterized in that, In step 1), the preparation of the reference drug solution from the tortoise shell glue reference material specifically involves: mixing the tortoise shell glue reference material with ammonium bicarbonate and sonicating it, adding trypsin for enzymatic hydrolysis, filtering, and taking the filtrate to obtain the reference drug solution.

3. The method for establishing the LCMS characteristic spectrum of tortoise shell glue and its preparations according to claim 2, characterized in that, In step 1), the mass-to-volume ratio of tortoise shell glue reference material to ammonium bicarbonate solution, and the mass-to-volume ratio of tortoise shell glue and its preparations to ammonium bicarbonate solution are both 1g:(200~1000)ml, the mass ratio of trypsin to tortoise shell glue and its preparations is 1:(2~4), and the concentration of trypsin is 0.5~2mg / ml; the power of the ultrasonic treatment is 300~500W, the frequency of the ultrasonic treatment is 30~50kHz, and the time of the ultrasonic treatment is 10~40min; in step 1), the mass concentration of tortoise shell polypeptide C1 in the reference solution is 1-5μg / ml.

4. The method for establishing the LCMS characteristic spectrum of tortoise shell glue and its preparations according to claim 1 or 2, characterized in that, In step 2), the chromatographic conditions of the liquid chromatography-mass spectrometry method are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; mobile phase A is 0.08-0.12 wt.% formic acid, and mobile phase B is acetonitrile, with mobile phase A and mobile phase B eluted in a gradient ratio by volume.

5. The method for establishing the LCMS characteristic spectrum of tortoise shell glue and its preparations according to claim 4, characterized in that, The gradient elution time is 20 minutes, and during the period from 0 to 12 minutes, the volume ratio of mobile phase A gradually increases from 2% to 10%, while the volume ratio of mobile phase B gradually decreases from 98% to 90%; during the period from 12 to 20 minutes, the volume ratio of mobile phase A gradually increases from 10% to 25%, while the volume ratio of mobile phase B gradually decreases from 90% to 75%; the chromatographic column is a chromatographic column with an inner diameter of 2.1 mm, a length of 100 mm, and a packing particle size of 1.7 μm; or a chromatographic column with an inner diameter of 2.1 mm, a length of 100 mm, and a packing particle size of 1.8 μm.

6. The method for establishing the LCMS characteristic spectrum of tortoise shell glue and its preparations according to claim 1 or 2, characterized in that, In the liquid chromatography-mass spectrometry method, the flow rate of the mobile phase is 0.1-0.3 mL per minute, the column temperature is 35-45℃, and the theoretical plate number calculated based on the C1 peak of the turtle-derived polypeptide is not less than 5000.

7. The method for establishing the LCMS characteristic spectrum of tortoise shell glue and its preparations according to claim 1 or 2, characterized in that, In step 2), the ion pairs in the liquid chromatography-mass spectrometry (LC-MS) method are set to the following mass-to-charge ratios for the parent ion and daughter ion: Peak 1 is m / z 427.2 (double charge) → 127.1 and m / z 427.2 (double charge) → 699.3; Peak 2 is m / z 523.8 (double charge) → 619.3 and m / z 523.8 (double charge) → 876.4; Peak 3 is m / z 568.7 (double charge) → 724.3 and m / z 568.7 (double charge) → 852.0; Peak 4 is m / Seven pairs of ion pairs: m / z 441.7 (double charge) → 528.3 and m / z 441.7 (double charge) → 629.3, peak 5 is m / z 626.3 (double charge) → 696.4 and m / z 626.3 (double charge) → 753.4, peak 6 is m / z 631.3 (double charge) → 546.4 and m / z 631.3 (double charge) → 921.4, peak 7 is m / z 758.3 (double charge) → 615.1 and m / z 758.3 (double charge) → 544.

1.

8. An LCMS characteristic spectrum of a tortoise shell glue and its preparations, characterized in that, The characteristic spectrum is a characteristic spectrum obtained by the method of any one of claims 1 to 7. The characteristic spectrum uses the turtle-derived polypeptide C with an ion mass-to-charge ratio of m / z 631.3 as the reference peak S, and has at least 6 characteristic peaks. The ion mass-to-charge ratios of the 6 characteristic peaks are m / z 427.2, m / z 523.8, m / z 568.7, m / z 441.7, m / z 626.3 and m / z 758.3, respectively, and the relative retention times of the characteristic peaks are all within ±15% of their respective specified values, which are 0.39, 0.42, 0.56, 0.58, 0.66 and 1.19, respectively.

9. The LCMS characteristic spectrum of the tortoise shell glue and its preparations according to claim 1, characterized in that, The characteristic spectrum exhibits at least seven characteristic peaks: peak 1 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.39 ± 15%; peak 2 has an ion mass-to-charge ratio of m / z 523.8 and a relative retention time of 0.42 ± 15%; peak 3 has an ion mass-to-charge ratio of m / z 568.7 and a relative retention time of 0.56 ± 15%; peak 4 has an ion mass-to-charge ratio of m / z 441.7 and a relative retention time of 0.58 ± 15%; peak 5 has an ion mass-to-charge ratio of m / z 626.3 and a relative retention time of 0.66 ± 15%; peak 6 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.39 ± 15%; peak 6 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.39 ± 15%; peak 7 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.42 ± 15%; peak 8 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.56 ± 15%; peak 9 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.58 ± 15%; peak 10 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.66 ± 15%; peak 11 has an ion mass-to-charge ratio of m / z 427.2 and a relative retention time of 0.39 ± 15%; peak 12 has an ion mass-to-charge ratio of m / The turtle-derived polypeptide C at m / z 631.3 has peak 6 as reference peak S and peak 7 at m / z 758.3 with a relative retention time of 1.19 ± 15%. The amino acid sequences of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, and peak 7 are: GPSGPEGPR, GAGEPPGSAGSK, GVQGAPGPQGPR, VGPTGPAGAR, GESGPAGPAGPAGAR, GIPGPAGPAGAAGPR, and GDGGPPGITGFPGASGR.

10. An application of LCMS feature map in the detection of tortoise shell glue, characterized in that, The LCMS feature map is the LCMS feature map as described in claim 8 or 9, or the LCMS feature map obtained by the establishment method described in any one of claims 1 to 7.

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